Spectacular advances in the ability to manipulate, fabricate and alter tiny subjects at the nanometer scale (Nanotechnology) have revolutionized material science in the past decade. The recent application of nanotechnology to life sciences has shown exciting promises in a wide range of disciplines. The development of laser-based technology (confocal microscope, laser tweezers, laser scissors, mutilphoton excitation confocal microscope, near field microscope, etc.) now allows functional imaging of living cells in thick tissues; the manipulation of single molecules, single organelles and single cells; the determination of the binding force and rate of interaction of DNA and other single molecules; the surgery of chromosomes and organelles in a living cell and the fabrication of miniature medical devices. The development of scanning probe microscopy (atomic force, scanning tunneling and electrochemical probe, near field microscopy) has enabled the manipulation of single molecules, the preparation of novel biochips and biosensors, and the measurement of physical and spectroscopic properties of single molecules in living cells. Our workgroup have succeeded in establishing state-of-the-art nanotechnology facilities (atomic force microscope, cellular force microscope, single fiber force station, real time confocal microscope, single molecule fluorescence microscope, and digital image analysis) to apply, adapt and develop bio-nanotechnology. These techniques are being applied to study single motors such as myosin and kinesin as well elastic proteins such a titin and nebulin, muscle filaments, cytoskeletal filaments, receptors in cellular membranes and cellular organelles such as myofibril, ribosome and chromatin. More specifically, our goals are: 1. Measuring the mechanical property and spectroscopy of single molecules, organelles and single cells. 2. Measuring the strength, speed and movement of interacting single molecules/organelles/cells. 3. Manipulating and altering intracellular organelles in living cells. 4. Fabricating and designing new instrumentation in nanotechnology. These direct measurement and manipulation of single molecule and filaments and organelles will provide unique and important insights of the events in the assembly and function of contractile machinery in muscle and nonmuscle cells. These studies will also reveal important engineering principles for designing tissues with prescribed mechanical properties. In the past year, we have successfully applied atomic force microscopy to image and measure the elastic properties of single monomeric protein, oligomeric protein and genetically engineered titin and nebulin molecules. We are also applying the single molecule fluorescence microscope to study the morphology and dynamic interactions of titin, nebulin, actin and myosin motors at the single molecule level. In this report year, we have made major progress in 1. Understanding how titin PEVK segment develops and modulates elasticity by correlating single molecule mechanical measurements with conformational states. 2. The development of an AFM that measure dynamics stiffness and force simultaneously in aqueous solutions. 3. The engineering of protein polymers based on selected PEVK modules to obtain defined elastic properties. These polymers also formed the basis for drug delivery hydrogels. Three manuscripts are at various stage of submission/preparation.